Aerosol-cloud interactions studied with a chemistry-climate model

This study aims at a comprehensive understanding of the effects of aerosol-cloud interactions and their effects on cloud properties and climate using the chemistry-climate model EMAC. In this study, CCN activation is regarded as the dominant driver in aerosol-cloud feedback loops in warm clouds. The CCN activation is calculated prognostically using two different cloud droplet nucleation parameterizations, the STN and HYB CDN schemes. Both CDN schemes account for size and chemistry effects on the droplet formation based on the same aerosol properties. The calculation of the solute effect (hygroscopicity) is the main difference between the CDN schemes. The kappa-method is for the first time incorporated into Abdul-Razzak and Ghan activation scheme (ARG) to calculate hygroscopicity and critical supersaturation of aerosols (HYB), and the performance of the modied scheme is compared with the osmotic coefficient model (STN), which is the standard in the ARG scheme. Reference simulations (REF) with the prescribed cloud droplet number concentration have also been carried out in order to understand the effects of aerosol-cloud feedbacks. In addition, since the calculated cloud coverage is an important determinant of cloud radiative effects and is influencing the nucleation process two cloud cover parameterizations (i.e., a relative humidity threshold; RH-CLC and a statistical cloud cover scheme; ST-CLC) have been examined together with the CDN schemes, and their effects on the simulated cloud properties and relevant climate parameters have been investigated. The distinct cloud droplet spectra show strong sensitivity to aerosol composition effects on cloud droplet formation in all particle sizes, especially for the Aitken mode. As Aitken particles are the major component of the total aerosol number concentration and CCN, and are most sensitive to aerosol chemical composition effect (solute effect) on droplet formation, the activation of Aitken particles strongly contribute to total cloud droplet formation and thereby providing different cloud droplet spectra. These different spectra influence cloud structure, cloud properties, and climate, and show regionally varying sensitivity to meteorological and geographical condition as well as the spatiotemporal aerosol properties (i.e., particle size, number, and composition). The changes responding to different CDN schemes are more pronounced at lower altitudes than higher altitudes. Among regions, the subarctic regions show the strongest changes, as the lower surface temperature amplifies the effects of the activated aerosols; in contrast, the Sahara desert, where is an extremely dry area, is less influenced by changes in CCN number concentration. The aerosol-cloud coupling effects have been examined by comparing the prognostic CDN simulations (STN, HYB) with the reference simulation (REF). Most pronounced effects are found in the cloud droplet number concentration, cloud water distribution, and cloud radiative effect. The aerosol-cloud coupling generally increases cloud droplet number concentration; this decreases the efficiency of the formation of weak stratiform precipitation, and increases the cloud water loading. These large-scale changes lead to larger cloud cover and longer cloud lifetime, and contribute to high optical thickness and strong cloud cooling effects. This cools the Earth's surface, increases atmospheric stability, and reduces convective activity. These changes corresponding to aerosol-cloud feedbacks are also differently simulated depending on the cloud cover scheme. The ST-CLC scheme is more sensitive to aerosol-cloud coupling, since this scheme uses a tighter linkage of local dynamics and cloud water distributions in cloud formation process than the RH-CLC scheme. For the calculated total cloud cover, the RH-CLC scheme simulates relatively similar pattern to observations than the ST-CLC scheme does, but the overall properties (e.g., total cloud cover, cloud water content) in the RH simulations are overestimated, particularly over ocean. This is mainly originated from the difference in simulated skewness in each scheme: the RH simulations calculate negatively skewed distributions of cloud cover and relevant cloud water, which is similar to that of the observations, while the ST simulations yield positively skewed distributions resulting in lower mean values than the RH-CLC scheme does. The underestimation of total cloud cover over ocean, particularly over the intertropical convergence zone (ITCZ) relates to systematic defficiency of the prognostic calculation of skewness in the current set-ups of the ST-CLC scheme.rnOverall, the current EMAC model set-ups perform better over continents for all combinations of the cloud droplet nucleation and cloud cover schemes. To consider aerosol-cloud feedbacks, the HYB scheme is a better method for predicting cloud and climate parameters for both cloud cover schemes than the STN scheme. The RH-CLC scheme offers a better simulation of total cloud cover and the relevant parameters with the HYB scheme and single-moment microphysics (REF) than the ST-CLC does, but is not very sensitive to aerosol-cloud interactions.

Interactions of gold nanoparticles with polymeric membranes

This thesis focuses on the interactions of nanoparticles with artificial membranes. The synthesis of the block copolymer poly(dimethylsiloxane)-block-poly(2-methyloxazoline) (PDMS-b-PMOXA) is described, as well as the formation of polymersomes in water. These polymersomes act as minimal cell models, consisting of an artificial bilayer membrane only, allowing the study of the interactions between nanoparticles and polymeric membranes. Both spherical and rod-shaped gold nanoparticles (AuNPs) were used in this study and they were characterized using light scattering (PCS), transmission electron microscopy (TEM), UV/Vis spectroscopy, and polarization anisotropy measurements. The polymer grafting on the spherical cores is asymmetric (shell asphericity) but is parallel to the inherent, due to polycrystallinity, core anisotropy, resulting in a characteristic scattering of the AuNPs in PCS.rnInteractions of polymersomes and AuNPs were investigated by PCS, cryo-TEM and UV/Vis. Three possible scenarios upon mixing of polymersomes and AuNPs can be distinguished by using only PCS: (i) no interactions between particles and vesicles, (ii) attachment of the particles to the outer side of the vesicles (decoration), and (iii) uptake of particles into the vesicles. It is shown that all three scenarios are possible, solely depending on the particle’s surface functionalization. In addition, it was revealed that the AuNPs need to be attached to the inner side of the membrane instead of diffusing freely within the vesicle. The present experimental findings essentially help with the understanding of the interactions of nanoparticles with membranes and show that the process of endocytosis can be attributed to physical processes only. rn

Stabilizing molecular self-assemblies via on-surface reactions

Für die Realisierung zukünftiger Technologien, wie z.B. molekulare Elektronik, werden Strategien benötigt, um funktionale Strukturen direkt auf Oberflächen zu erzeugen. Für die Bewältigung dieser Aufgabe ist die molekulare Selbstanordnung ein äußerst vielversprechender Bottom-up-Ansatz. Hierbei ist eine der größten Herausforderungen das Zusammenspiel aus intramolekularer Wechselwirkung und der Wechselwirkung zwischen Substrat und Molekülen in ein Gleichgewicht zu bringen. Da jedoch die wirkenden Kräfte der molekularen Selbstanordnung ausschließlich reversibler Natur sind, ist eine langfristige Stabilität fragwürdig. Somit ist die kovalente Verknüpfung der gebildeten Strukturen durch Reaktionen direkt auf der Oberfläche unerlässlich, um die Stabilität der Strukturen weiter zu erhöhen. Hierzu stellt die vorliegende Arbeit eine ausführliche Studie zu molekularer Selbstanordnung und der zielgerichteten Modifikation ebensolcher Strukturen dar. Durch den Einsatz von hochauflösender Rasterkraftmikroskopie im Ultrahochvakuum, welche es erlaubt einzelne Moleküle auf Nichtleitern abzubilden, wurde der maßgebliche Einfluss von Ankerfunktionalitäten auf den Prozess der molekularen Selbstanordnung gezeigt. Des Weiteren konnte die Stabilität der selbst angeordneten Strukturen durch neue Oberflächenreaktionskonzepte entschieden verbessert werden. Der Einfluss von Ankerfunktionen, die elektrostatische Wechselwirkung zwischen Molekül und Substrat vermitteln, auf den Strukturbildungsprozess der molekularen Selbstanordnung wird eingehend durch den Vergleich eines aromatischen Moleküls und seines vierfach chlorierten Derivates gezeigt. Für diese beiden Moleküle wurde ein deutlich unterschiedliches Verhalten der Selbstanordnung beobachtet. Es wird gezeigt, dass die Fähigkeit zur Bildung selbst angeordneter, stabiler Inseln entscheidend durch die Substituenten und die Abmessungen des Moleküls beeinflusst wird. Auch wird in dieser Arbeit die erste photochemische Reaktion organischer Moleküle auf einem Isolator gezeigt. Qualitative und quantitative Ergebnisse liefern ein detailliertes Bild darüber, wie die Abmessungen des Substratgitters die Richtung der Reaktion gezielt beeinflussen. Des Weiteren wird ein allgemeines Konzept zur selektiven Stabilisierung selbstangeordneter Molekülstrukturen durch den kontrollierten Transfer von Elektronen präsentiert. Durch die gezielte Steuerung der Menge an Dotierungsatomen wird die Desorptionstemperatur der molekularen Inseln signifikant erhöht und das Desorptionsverhalten der Inseln entschieden verändert. Diese Arbeit präsentiert somit erfolgreich durchgeführte Strategien um den Prozess der molekularen Selbstanordnung zu steuern, sowie entscheidende Mechanismen um die Stabilisierung und Modifizierung von selbst angeordneten Strukturen zu gewährleisten.

The role of the interplay between polymer architecture and bacterial surface properties on the microbial adhesion to polyoxazoline-based ultrathin films

Surface platforms were engineered from poly(L-lysine)-graft-poly(2-methyl-2-oxazoline) (PLL-g-PMOXA) copolymers to study the mechanisms involved in the non-specific adhesion of Escherichia coli (E. coli) bacteria. Copolymers with three different grafting densities (PMOXA chains/Lysine residue of 0.09, 0.33 and 0.56) were synthesized and assembled on niobia (Nb O ) surfaces. PLL-modified and bare niobia surfaces served as controls. To evaluate the impact of fimbriae expression on the bacterial adhesion, the surfaces were exposed to genetically engineered E. coli strains either lacking, or constitutively expressing type 1 fimbriae. The bacterial adhesion was strongly influenced by the presence of bacterial fimbriae. Non-fimbriated bacteria behaved like hard, charged particles whose adhesion was dependent on surface charge and ionic strength of the media. In contrast, bacteria expressing type 1 fimbriae adhered to the substrates independent of surface charge and ionic strength, and adhesion was mediated by non-specific van der Waals and hydrophobic interactions of the proteins at the fimbrial tip. Adsorbed polymer mass, average surface density of the PMOXA chains, and thickness of the copolymer films were quantified by optical waveguide lightmode spectroscopy (OWLS) and variable-angle spectroscopic ellipsometry (VASE), whereas the lateral homogeneity was probed by time-of-flight secondary ion mass spectrometry (ToF-SIMS). Streaming current measurements provided information on the charge formation of the polymer-coated and the bare niobia surfaces. The adhesion of both bacterial strains could be efficiently inhibited by the copolymer film only with a grafting density of 0.33 characterized by the highest PMOXA chain surface density and a surface potential close to zero.

Sensitive and selective detection of free FXIII activation peptide: a potential marker of acute thrombotic events

Coagulation factor XIII (FXIII) stabilizes fibrin fibers and is therefore a major player in the maintenance of hemostasis. FXIII is activated by thrombin resulting in cleavage and release of the FXIII activation peptide (AP-FXIII). The objective of this study was to characterize the released AP-FXIII and determine specific features that may be used for its specific detection. We analyzed the structure of bound AP-FXIII within the FXIII A-subunit and interactions of AP-FXIII by hydrogen bonds with both FXIII A-subunit monomers. We optimized our previously developed AP-FXIII ELISA by using 2 monoclonal antibodies. We determined high binding affinities between the antibodies and free AP-FXIII and demonstrated specific binding by epitope mapping analyses with surface plasmon resonance and enzyme-linked immunosorbent assay. Because the structure of free AP-FXIII had been characterized so far by molecular modeling only, we performed structural analysis by nuclear magnetic resonance. Recombinant AP-FXIII was largely flexible both in plasma and water, differing significantly from the rigid structure in the bound state. We suggest that the recognized epitope is either occluded in the noncleaved form or possesses a structure that does not allow binding to the antibodies. On the basis of our findings, we propose AP-FXIII as a possible new marker for acute thrombotic events.

The contribution of surface residues to membrane binding and ligand transfer by the -tocopherol transfer protein ( -TTP)

Previous work has shown that the -tocopherol transfer protein ( -TTP) can bind to vesicular or immobilized phospholipid membranes. Revealing the molecular mechanisms by which -TTP associates with membranes is thought to be critical to understanding its function and role in the secretion of tocopherol from hepatocytes into the circulation. Calculations presented in the Orientations of Proteins in Membranes database have provided a testable model for the spatial arrangement of -TTP and other CRAL-TRIO family proteins with respect to the lipid bilayer. These calculations predicted that a hydrophobic surface mediates the interaction of -TTP with lipid membranes. To test the validity of these predictions, we used site-directed mutagenesis and examined the substituted mutants with regard to intermembrane ligand transfer, association with lipid layers and biological activity in cultured hepatocytes. Substitution of residues in helices A8 (F165A and F169A) and A10 (I202A, V206A and M209A) decreased the rate of intermembrane ligand transfer as well as protein adsorption to phospholipid bilayers. The largest impairment was observed upon mutation of residues that are predicted to be fully immersed in the lipid bilayer in both apo (open) and holo (closed) conformations such as Phe165 and Phe169. Mutation F169A, and especially F169D, significantly impaired -TTP-assisted secretion of -tocopherol outside cultured hepatocytes. Mutation of selected basic residues (R192H, K211A, and K217A) had little effect on transfer rates, indicating no significant involvement of nonspecific electrostatic interactions with membranes.

T-cadherin is present on endothelial microparticles and is elevated in plasma in early atherosclerosis

The presence of endothelial cell (EC)-derived surface molecules in the circulation is among hallmarks of endothelial activation and damage in vivo. Previous investigations suggest that upregulation of T-cadherin (T-cad) on the surface of ECs may be a characteristic marker of EC activation and stress. We investigated whether T-cad might also be shed from ECs and in amounts reflecting the extent of activation or damage.

Plasma membrane repair and cellular damage control: the annexin survival kit

Plasmalemmal injury is a frequent event in the life of a cell. Physical disruption of the plasma membrane is common in cells that operate under conditions of mechanical stress. The permeability barrier can also be breached by chemical means: pathogens gain access to host cells by secreting pore-forming toxins and phospholipases, and the host's own immune system employs pore-forming proteins to eliminate both pathogens and the pathogen-invaded cells. In all cases, the influx of extracellular Ca(2+) is being sensed and interpreted as an "immediate danger" signal. Various Ca(2+)-dependent mechanisms are employed to enable plasma membrane repair. Extensively damaged regions of the plasma membrane can be patched with internal membranes delivered to the cell surface by exocytosis. Nucleated cells are capable of resealing their injured plasmalemma by endocytosis of the permeabilized site. Likewise, the shedding of membrane microparticles is thought to be involved in the physical elimination of pores. Membrane blebbing is a further damage-control mechanism, which is triggered after initial attempts at plasmalemmal resealing have failed. The members of the annexin protein family are ubiquitously expressed and function as intracellular Ca(2+) sensors. Most cells contain multiple annexins, which interact with distinct plasma membrane regions promoting membrane segregation, membrane fusion and--in combination with their individual Ca(2+)-sensitivity--allow spatially confined, graded responses to membrane injury.

Fine-tuning of substrate architecture and surface chemistry promotes muscle tissue development

Tissue engineering has been increasingly brought to the scientific spotlight in response to the tremendous demand for regeneration, restoration or substitution of skeletal or cardiac muscle after traumatic injury, tumour ablation or myocardial infarction. In vitro generation of a highly organized and contractile muscle tissue, however, crucially depends on an appropriate design of the cell culture substrate. The present work evaluated the impact of substrate properties, in particular morphology, chemical surface composition and mechanical properties, on muscle cell fate. To this end, aligned and randomly oriented micron (3.3±0.8 μm) or nano (237±98 nm) scaled fibrous poly(ε-caprolactone) non-wovens were processed by electrospinning. A nanometer-thick oxygen functional hydrocarbon coating was deposited by a radio frequency plasma process. C2C12 muscle cells were grown on pure and as-functionalized substrates and analysed for viability, proliferation, spatial orientation, differentiation and contractility. Cell orientation has been shown to depend strongly on substrate architecture, being most pronounced on micron-scaled parallel-oriented fibres. Oxygen functional hydrocarbons, representing stable, non-immunogenic surface groups, were identified as strong triggers for myotube differentiation. Accordingly, the highest myotube density (28±15% of total substrate area), sarcomeric striation and contractility were found on plasma-coated substrates. The current study highlights the manifold material characteristics to be addressed during the substrate design process and provides insight into processes to improve bio-interfaces.

Acquisition of a multifunctional IgA+ plasma cell phenotype in the gut

The largest mucosal surface in the body is in the gastrointestinal tract, a location that is heavily colonized by microbes that are normally harmless. A key mechanism required for maintaining a homeostatic balance between this microbial burden and the lymphocytes that densely populate the gastrointestinal tract is the production and transepithelial transport of poly-reactive IgA (ref. 1). Within the mucosal tissues, B cells respond to cytokines, sometimes in the absence of T-cell help, undergo class switch recombination of their immunoglobulin receptor to IgA, and differentiate to become plasma cells. However, IgA-secreting plasma cells probably have additional attributes that are needed for coping with the tremendous bacterial load in the gastrointestinal tract. Here we report that mouse IgA(+) plasma cells also produce the antimicrobial mediators tumour-necrosis factor-α (TNF-α) and inducible nitric oxide synthase (iNOS), and express many molecules that are commonly associated with monocyte/granulocytic cell types. The development of iNOS-producing IgA(+) plasma cells can be recapitulated in vitro in the presence of gut stroma, and the acquisition of this multifunctional phenotype in vivo and in vitro relies on microbial co-stimulation. Deletion of TNF-α and iNOS in B-lineage cells resulted in a reduction in IgA production, altered diversification of the gut microbiota and poor clearance of a gut-tropic pathogen. These findings reveal a novel adaptation to maintaining homeostasis in the gut, and extend the repertoire of protective responses exhibited by some B-lineage cells.

Enamel matrix protein adsorption to root surfaces in the presence or absence of human blood

Background: The clinical use of an enamel matrix derivative (EMD) has been shown to promote formation of new cementum, periodontal ligament (PDL), and bone and to significantly enhance the clinical outcomes after regenerative periodontal surgery. It is currently unknown to what extent the bleeding during periodontal surgery may compete with EMD adsorption to root surfaces. The aim of this study is to evaluate the effect of blood interactions on EMD adsorption to root surfaces mimicking various clinical settings and to test their ability to influence human PDL cell attachment and proliferation. Methods: Teeth extracted for orthodontic reasons were subjected to ex vivo scaling and root planing and treated with 24% EDTA, EMD, and/or human blood in six clinically related settings to determine the ability of EMD to adsorb to root surfaces. Surfaces were analyzed for protein adsorption via scanning electron microscopy and immunohistochemical staining with an anti-EMD antibody. Primary human PDL cells were seeded on root surfaces and quantified for cell attachment and cell proliferation. Results: Plasma proteins from blood samples altered the ability of EMD to adsorb to root surfaces on human teeth. Samples coated with EMD lacking blood demonstrated a consistent even layer of EMD adsorption to the root surface. In vitro experiments with PDL cells demonstrated improved cell attachment and proliferation in all samples coated with EMD (irrespective of EDTA) when compared to samples containing human blood. Conclusion: Based on these findings, it is advised to minimize blood interactions during periodontal surgeries to allow better adsorption of EMD to root surfaces.

Interaction Between Nitrous Oxide, Sevoflurane, and Opioids: A Response Surface Approach

BACKGROUND:: The interaction of sevoflurane and opioids can be described by response surface modeling using the hierarchical model. We expanded this for combined administration of sevoflurane, opioids, and 66 vol.% nitrous oxide (N2O), using historical data on the motor and hemodynamic responsiveness to incision, the minimal alveolar concentration, and minimal alveolar concentration to block autonomic reflexes to nociceptive stimuli, respectively. METHODS:: Four potential actions of 66 vol.% N2O were postulated: (1) N2O is equivalent to A ng/ml of fentanyl (additive); (2) N2O reduces C50 of fentanyl by factor B; (3) N2O is equivalent to X vol.% of sevoflurane (additive); (4) N2O reduces C50 of sevoflurane by factor Y. These four actions, and all combinations, were fitted on the data using NONMEM (version VI, Icon Development Solutions, Ellicott City, MD), assuming identical interaction parameters (A, B, X, Y) for movement and sympathetic responses. RESULTS:: Sixty-six volume percentage nitrous oxide evokes an additive effect corresponding to 0.27 ng/ml fentanyl (A) with an additive effect corresponding to 0.54 vol.% sevoflurane (X). Parameters B and Y did not improve the fit. CONCLUSION:: The effect of nitrous oxide can be incorporated into the hierarchical interaction model with a simple extension. The model can be used to predict the probability of movement and sympathetic responses during sevoflurane anesthesia taking into account interactions with opioids and 66 vol.% N2O.

Effects of MASP-1 of the complement system on activation of coagulation factors and plasma clot formation

Background Numerous interactions between the coagulation and complement systems have been shown. Recently, links between coagulation and mannan-binding lectin-associated serine protease-1 (MASP-1) of the complement lectin pathway have been proposed. Our aim was to investigate MASP-1 activation of factor XIII (FXIII), fibrinogen, prothrombin, and thrombin-activatable fibrinolysis inhibitor (TAFI) in plasma-based systems, and to analyse effects of MASP-1 on plasma clot formation, structure and lysis. Methodology/Principal Findings We used a FXIII incorporation assay and specific assays to measure the activation products prothrombin fragment F1+2, fibrinopeptide A (FPA), and activated TAFI (TAFIa). Clot formation and lysis were assessed by turbidimetric assay. Clot structure was studied by scanning electron microscopy. MASP-1 activated FXIII and, contrary to thrombin, induced FXIII activity faster in the Val34 than the Leu34 variant. MASP-1-dependent generation of F1+2, FPA and TAFIa showed a dose-dependent response in normal citrated plasma (NCP), albeit MASP-1 was much less efficient than FXa or thrombin. MASP-1 activation of prothrombin and TAFI cleavage were confirmed in purified systems. No FPA generation was observed in prothrombin-depleted plasma. MASP-1 induced clot formation in NCP, affected clot structure, and prolonged clot lysis. Conclusions/Significance We show that MASP-1 interacts with plasma clot formation on different levels and influences fibrin structure. Although MASP-1-induced fibrin formation is thrombin-dependent, MASP-1 directly activates prothrombin, FXIII and TAFI. We suggest that MASP-1, in concerted action with other complement and coagulation proteins, may play a role in fibrin clot formation.

Echinococcus multilocularis phosphoglucose isomerase (EmPGI): a glycolytic enzyme involved in metacestode growth and parasite-host cell interactions

In Echinococcus multilocularis metacestodes, the surface-associated and highly glycosylated laminated layer, and molecules associated with this structure, is believed to be involved in modulating the host-parasite interface. We report on the molecular and functional characterisation of E. multilocularis phosphoglucose isomerase (EmPGI), which is a component of this laminated layer. The EmPGI amino acid sequence is virtually identical to that of its homologue in Echinococcus granulosus, and shares 64% identity and 86% similarity with human PGI. Mammalian PGI is a multi-functional protein which, besides its glycolytic function, can also act as a cytokine, growth factor and inducer of angiogenesis, and plays a role in tumour growth, development and metastasis formation. Recombinant EmPGI (recEmPGI) is also functionally active as a glycolytic enzyme and was found to be present, besides the laminated layer, in vesicle fluid and in germinal layer cell extracts. EmPGI is released from metacestodes and induces a humoral immune response in experimentally infected mice, and vaccination of mice with recEmPGI renders these mice more resistant towards secondary challenge infection, indicating that EmPGI plays an important role in parasite development and/or in modulating the host-parasite relationship. We show that recEmPGI stimulates the growth of isolated E. multilocularis germinal layer cells in vitro and selectively stimulates the proliferation of bovine adrenal cortex endothelial cells but not of human fibroblasts and rat hepatocytes. Thus, besides its role in glycolysis, EmPGI could also act as a factor that stimulates parasite growth and potentially induces the formation of novel blood vessels around the developing metacestode in vivo.

Structural basis for the sheddase function of human meprin β metalloproteinase at the plasma membrane

Ectodomain shedding at the cell surface is a major mechanism to regulate the extracellular and circulatory concentration or the activities of signaling proteins at the plasma membrane. Human meprin β is a 145-kDa disulfide-linked homodimeric multidomain type-I membrane metallopeptidase that sheds membrane-bound cytokines and growth factors, thereby contributing to inflammatory diseases, angiogenesis, and tumor progression. In addition, it cleaves amyloid precursor protein (APP) at the β-secretase site, giving rise to amyloidogenic peptides. We have solved the X-ray crystal structure of a major fragment of the meprin β ectoprotein, the first of a multidomain oligomeric transmembrane sheddase, and of its zymogen. The meprin β dimer displays a compact shape, whose catalytic domain undergoes major rearrangement upon activation, and reveals an exosite and a sugar-rich channel, both of which possibly engage in substrate binding. A plausible structure-derived working mechanism suggests that substrates such as APP are shed close to the plasma membrane surface following an "N-like" chain trace.